title: Association of acyl-CoA synthetase-1 with GLUT4-containing vesicles.
abstract: GLUT4, the glucose transporter present in insulin-sensitive tissues, resides in intracellular vesicular structures and translocates to the cell surface in response to insulin. In an attempt to identify proteins present in these structures, GLUT4-enriched vesicles prepared from rat adipocytes treated with or without insulin were prepared by sucrose velocity gradient centrifugation and immunoadsorbed with anti-GLUT4 antibody. We report here the sequence identification by high performance liquid chromatography-ion trap mass spectrometry of a p75 protein band, long chain acyl-CoA synthetase-1, specifically present in immunoadsorbed GLUT4-containing vesicles but not in vesicles adsorbed by nonimmune serum. Acyl-CoA synthetase activity detected in GLUT4-enriched vesicles prepared by gradient centrifugation from insulin-treated adipocytes was decreased to about the same extent as GLUT4 protein. Additionally, immunoadsorbed GLUT4 vesicles were found to catalyze palmitoylation of proteins when incubated with labeled palmitate, a pathway that requires palmitate esterification with CoA. These data indicate that the insulin-sensitive membrane compartment that sequesters GLUT4 in fat cells contains long chain acyl-CoA synthetase-1 and its product fatty acyl-CoA, shown previously to be required for budding and fusion in membrane trafficking processes.
authors.affiliation: Program in Molecular Medicine and the Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605, USA.
authors.forename: M W
authors.initials: MW
authors.lastname: Sleeman
authors.forename: N P
authors.initials: NP
authors.lastname: Donegan
authors.forename: R
authors.initials: R
authors.lastname: Heller-Harrison
authors.forename: W S
authors.initials: WS
authors.lastname: Lane
authors.forename: M P
authors.initials: MP
authors.lastname: Czech
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D017667:Adipocytes / Q000201:enzymology* / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D003066:Coenzyme A Ligases / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D008297:Male; D008561:Membrane Fusion; D008854:Microscopy, Electron; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
chemical_list: D000214:Acyl Coenzyme A; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C495694:Slc2a4 protein, rat; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase